The basic antibiotics of the .beta.-lactam type are principally obtained by fermentation. Fungi of the genus Penicillium and Cephalosporium (Acremonium) are used for the production of raw material for .beta.-lactam antibiotics as penicillin G, penicillin V and cephalosporin C. These fermentation products, also referred to as PenG, PenV and CefC, respectively, are the starting materials for nearly all currently marketed penicillins and cephalosporins. In general the acyl group at the 6-amino of the penicillin nucleus or at the 7-amino position of the cephalosporin nucleus is referred to as `side chain`, the corresponding acid as `side chain acid`. The side chains of PenG, PenV and CefC are phenylacetyl, phenoxy-acetyl and aminoadipyl, respectively. The side chains are removed by cleavage of an amide linkage (deacylation), resulting in 6-aminopenicillanic acid (6-APA) in case of the penicillin molecules and 7-aminocephalosporanic acid (7-ACA) in case of the cephalosporin molecule. In this respect also phenylacetyl-7-aminodesacetoxycepha-losporanic acid (CefG) should be mentioned as a precursor of 7-ADCA, although CefG is not a fermentation product. CefG is usually produced chemically from Penicillin G.
In order to obtain .beta.-lactam compounds with an altered activity spectrum, an increased resistance against .beta.-lactanases or improved clinical performance of .beta.-lactam compounds, 6-APA, 7-ACA and 7-ADCA are used as starting points for synthetic manipulation to produce the various penicillins and cephalosporins of choice. At present these semisynthetic penicillins and cephalosporins form by far the most important market of .beta.-lactam antibiotics.
The production of semisynthetic .beta.-lactam products requires the deacylation of the penicillins and cephalosporins produced from fermentation. Although rather efficient chemical routes are available for the deacylation (J. Verweij & E. de Vroom, Recl. Trav. Chim. Pays-Bas 112 (1993) 66-81), nowadays the enzymatic route is preferred in view of the high energy and solvents cost together with some environmental problems associated with the chemical route (Dunnill, P., Immobilised Cell and Enzyme Technology. Philos, Trans. R. Soc. London B290 (1980) 409-420). The enzymes which may accomplish the deacylation of .beta.-lactam compounds are classified as hydrolases based on the chemical reaction they catalyse. However, those hydrolases which are particularly useful in the deacylation is of .beta.-lactam compounds are usually referred to in the art as `acylases` or `amidases`. These denominations as used in this specification have the same meaning. In connection with .beta.-lactam antibiotics these acylases usually are further specified as `.beta.-lactam acylases` as not all amidases accept a .beta.-lactam nucleus as an acceptor/donor moiety for the acyl group. According to the literature several types of .beta.-lactam acylases may be envisaged, based on their substrate specificity and molecular structure (B. S. Deshpande et al., World J. Microbiology & Biotechnology 10 (1994) 129-138).
Acylase, Nomenclature & Classification
Classification according to Specificity.
The substrate specificity of the acylase is determined by a side chain binding pocket at the enzyme which recognizes the side chain moiety of .beta.-lactam molecules. In general, the acylases are not very specific for the moiety adjacent to the nitrogen atom of the amide group (this might be a cephem group, a penem group, an amino acid, sugars, etc. (J. G. Shewale et al., Process Biochemistry International, Jun. 1990, 97-103). In case of the Penicillin G acylases (Benzylpenicillin amidohydrolase, also named Penicillin amidase; EC 3.5.1.11) this acyl moiety must be very hydrophobic and is preferably phenylacetyl or (short) alkyl. Penicillin G acylase is used commercially to hydrolyse PenG or CefG to phenylacetic-acid and 6-APA or 7-ADCA, respectively the most important intermediates for the industrial production of semi-synthetic penicillins and cephalosporins. Beside these major applications other have been reported for these enzymes such as blocking/deblocking of sensitive groups in organic synthesis and peptide chemistry, stereospecific conversions, optical resolution of phenylglycine, deesterification of carbinols, acylation of mono-bactams etc. In the various applications the enzyme may be used C. either in its native state or as immobilised preparation. Microbial whole cells containing the enzyme activity have also been used either as cell suspension or as immobilised cell preparation.
Examples of substrates which are not hydrolyzed by Penicillin G acylases are those with charged acyl moieties such as dicarboxylic acids: succinyl, glutaryl, adipyl and also amino-adipyl, the side-chain of CefC.
Penicillin V acylases are highly specific for phenoxyacetyl, while ampicillin acylase prefers D-phenylglycine as a side chain. Glutaryl-acylases deacylate glutaryl-7-ACA, which is prepared from CefC after enzymatic deamidation of the side chain with D-amino acid oxidase followed by chemical decarboxylation of the formed ketoadipyl derivative with peroxide, which is produced in the first step. Moreover some of these acylases have been reported to be capable of hydrolyzing cephalosporins (including the desacetoxy-derivative) with succinyl, glutaryl and adipyl as an acyl moiety and even in one case CefC to a very limited degree (for a review see EP-A-322032, Merck). So far these acylases have only been found in Pseudomonas species, and in certain strains of Bacillus megaterium and Arthrobacter viscosus.
Classification based on structural properties of the enzymes.
Apart from their specificities acylases may also be classified based on molecular aspects (V. K. sudhakaran et al., Process Biochemistry 27 (1992) 131-143):
Type-I acylases are specific for Penicillin V. These enzymes are composed of four identical subunits, each having a molecular weight of 35 kDa. PA1 Type-II acylases all share a common molecular structure: these enzymes are heterodimers composed of a small subunit (.alpha.; 16-26 kDa) and a large subunit (.beta.; 54-66 kDa). With respect to the substrate specificity, Type-II acylases may be further divided into two groups: PA1 Type III acylases are the Ampicillin acylases which have been reported to be dimers consisting of two identical subunits with a molecular weight of 72 kDa. PA1 reactions can be performed stereospecifically; PA1 reactants do not require side chain protection such as silylation; PA1 less need for organic solvents, i.e. an organic solvent such as methylene chloride can be omitted which reduces environmental problems; PA1 compared to the chemical route usually less steps are required; PA1 neither extreme temperatures nor pressures required; PA1 usually lower content of byproducts. PA1 reaction medium: pH, ionic strength, temperature, organic solvents, etc.; PA1 enzyme stability with respect to process conditions; PA1 reactant stability; PA1 catalytic activity of the enzyme. PA1 a substitution at one or more selected sites of the positions corresponding to A139 to A152, B20 to B27, B31, B32, B49 to B52, B56, B57, B65 to B72, B154 to B157, B173 to B179, B239 to B241, B250 to B263, B379 to B387, B390, B455, B474 to B480 in Alcaligenes faecalis Penicillin G acylase or its pre- or preproenzyme; and PA1 an altered substrate specificity or altered specific activity relative to the corresponding wild-type unsubstituted Penicillin G acylase. Preferably, said isolated mutant prokaryotic Penicillin G acylase is originated from Alcaligenes faecalis. PA1 culturing a microorganism host strain transformed with an expression vector comprising a nucleic acid sequence encoding a mutant acylase enzyme as defined above, whereby said mutant acylase is produced; and PA1 isolating said acylase.
Type-IIA acylases comprise the Penicillin G acylases; PA2 Type-IIB acylases comprise the Glutaryl acylases.
Benefits of Protein Encineerina with Respect to Screening/Chemical Modification
Enzymes with improved properties can be developed or found in several ways, for example, by classical screening methods, by chemical modification of existing proteins, or by using modern genetic and protein-engineering techniques.
Screening for organisms or microorganisms that display the desired enzymatic activity, can be performed, for example, by isolating and purifying the enzyme from a microorganism or from a culture supernatant of such microorganisms, determining its biochemical properties and checking whether these biochemical properties meet the demands for application. The present collection of acylases results from intensive screening programs. .beta.-lactam acylase activity has been found in many microorganisms such as fungi, yeast, actinomycetes and bacteria.
If the identified enzyme cannot be obtained from its natural producing organism, recombinant-DNA techniques may be used to isolate the gene encoding the enzyme, express the gene in another organism, isolate and purify the expressed enzyme and test whether it is suitable for the intended application.
Modification of existing enzymes can be achieved inter alia by chemical modification methods. In general, these methods are too unspecific in that they modify all accessible residues with common side chains or they are dependent on the presence of suitable amino acids to be modified, and often they are unable to modify amino acids difficult to reach, unless the enzyme molecule is unfolded. In addition chemical modification require Additional processing steps and chemicals to prepare the enzyme. Enzyme modification through mutagenesis of the encoding gene does not suffer from the problems mentioned above, and therefore is thought to be superior.
Moreover the choice for an acylase, subsequent construction and selection of high-yielding penicillin acylase-producing strains and the development of an industrial process for isolation and immobilisation, is a laborious process. In general for production and subsequent formulation of the mutants the wild type protocols can be followed. Therefore, once such a process has been developed succesfully for a certain acylase it is very attractive to broaden the application of the acylase of choice instead of continuing the screening for enzymes from other sources. Therefore enzyme modification through mutagenesis of the encoding wild type gene is thought to be superior to screening especially when small adaptation of the properties of the enzyme are required. Desired properties may include altered specificity, altered specific activity for a certain substrate, altered pH dependence or altered stability. Mutagenesis can be achieved either by random mutagenesis or by site-directed mutagenesis.
Random mutagenesis, by treating whole microorganisms with chemical mutagens or with mutagenizing radiation, may of course result in modified enzymes, but then strong selection protocols are necessary to search for mutants having the desired properties. Higher probability of isolating desired mutant enzymes by random mutagenesis can be achieved by cloning the encoding enzyme, mutagenizing it in vitro or in vivo and expressing the encoded enzyme by recloning of the mutated gene in a suitable host cell. Also in this case suitable biological selection protocols must be available in order to select the desired mutant enzymes.
Site-directed mutagenesis (SDM) is the most specific way of obtaining modified enzymes, enabling specific substitution of one or more amino acids by any other desired amino acid.
The conversion of .beta.-lactam intermediates to the desired semi-synthetic antibiotics may be performed chemically and enzymatically. If a suitable enzyme is available the enzymatic route is preferred because:
Synthetic manipulation to produce the various penicillins and cephalosporins of choice basically starts from 6-APA, 7-ACA and 7-ADCA, respectively.
The enzymatic conversion takes advantage of the fact that any enzymatic reaction is reversible, if the correct conditions are applied. The importance of such applications has been highlighted in previous reviews. The literature gives several examples of the application of penicillin acylases in biosynthetic routes (J. G. Shewale et Al., Process Biochemistry International, June 1990, 97-103). Acyl derivatives of 6-APA, 7-ADCA, 7-ACA, 3-amino-4-.alpha.-methyl monobactamic acid and peptides have been prepared with side-chain moieties of varying structure. Besides 6-APA and 7-ADCA, penicillin acylase is used in the formation of antibiotic intermediates such as 6-amino-2,2-dimethyl-3-(tetrazol-5-yl) penam, methyl-6-aminopenicillate, 3-methyl-7-amino-3-cephem-4-carboxylic acid and 3-amino nocardic acid. The hydrolytic raction is catalysed at alkaline pH (7.5-8.5) while at acidic or neutral pH (4.0-7.0) it promotes acylation reactions.
Various factors affect the performance of an acylase in bioconversion processes:
Except reactant stability which is not an enzyme property, the other factors may be a target for enzyme modification via protein engineering.
Various of these factors have been explored in order to make biosynthesis processes economically viable. Methylesters which are superior acyl donors as compared to free acids of side chain acids have been used in the reaction. The equilibrium of the reaction has been shifted in favour of acylation by changing the water activity around the enzyme molecule with certain solvents. E.g. polyethyleneglycol, methanol, ethanol, propanol, butanol, and acetone are used in enhancing the yield of penicillin G, penicillin V and ampicillin.
Acylation reactions especially with 6-APA, 7-ADCA and 7-ACA generate antibiotics which are clinically important. However, the reaction needs to be monitored under strict kinetically controlled parameters. Although in some articles it was speculated that protein-engineering tools might be explored to obtain tailored enzyme molecules giving semisynthetic penicillins and cephalopsorins at a yield competing with existing chemical processes, there was no teaching whatsoever neither how this should be carried out, nor which enzymes should be engineered, or which amino acid residues should be substituted, nor any relation between the kind of substitution and the desired substrate.
The synthetic potential of a given penicillin acylase is limited due to the specificity of the enzyme. Therefore, there is a substantial interest in developing enzymes which are highly efficient in deacylation/acylation reactions to producedesired chemical entities. Of particular interest are the enzymatic deacylation of .beta.-lactams (especially PenG, PenV, CefC, and derivatives thereof) to 6-APA and 7-ACA and derivatives, and the acylation of the latter compounds to produce semi-synthetic pencillins and cephalosporins of interest. In addition increased activity on more polar side chains or charged side chains such as succinyl, glutaryl or adipyl is desired. In particular, it is of major importance to dispose of an efficient enzyme which is capable of catalyzing the conversion of CefC (and derivatives) to 7-ACA (and derivatives).
Theoretical Aspects of the Application of Enzymes in Synthesis
Penicillin G acylases are hydrolases which catalyse the deacylation of various .beta.-lactam compounds. Moreover as enzymes catalyse reactions in both directions, these acylases may also be used as a transferase to catalyse the synthesis of condensation products such as .beta.-lactam antibiotics, peptides, oligosaccharides or glycerides. Enzyme catalysed synthesis may be carried out either as an equilibrium controlled or as a kinetically controlled reaction.
In an equilibrium controlled process the enzyme only accelerates the rate at which the thermodynamic equilibrium is established. The kinetic properties of the enzyme do not influence the equilibrium concentrations. However, the thermodynamic equilibrium is dependent on reaction conditions such as pH, temperature, ionic strength, or solvent composition. Often the conditions which favour the shift of the thermodynamic equilibrium in such a way that an optimal yield of the desired product is obtained are usually not optimal for the performance of the enzyme. In such cases enzyme engineering may be desired to adapt the enzymes to conditions which are closer to the thermodynamic optimum of the reaction. In this aspect properties such as stability, temperature optimum and pH optimum may be useful targets.
In kinetically controlled reactions conditions are chosen in such way that a considerable accumulation occurs of the desired product during the reaction under non-equilibrium conditions. In this case besides the already mentioned parameters also the kinetic properties of the enzyme are an important factor in obtaining yields which can compete favourably with existing chemical processes.
The kinetics of Penicillin G acylase. are consistent with catalysis proceeding via an acyl-enzyme intermediate. This intermediate plays a key role in the enzyme mechanism as is depicted in FIG. 1. In this scheme the acylase acts as a hydrolase where the acyl group is transferred to water, or as a transterase where the acyl transfer from an activated substrate to a nucleophile is catalyzed. The chemical entities are represented by general formulas. The nature of the chemical entities X and Y in compound X--CO--NH--Y which are accepted as a substrate by a particular acylase is determined by the specificity of that acylase. X represents the side chain, while Y represents the acyl acceptor group. For instance, for the deacylation of PenG, X--CO-- represents the phenyl-acetyl side chain and --NH--Y represents 6-aminopenicillic acid. Given a certain enzymatic mechanism the specificity is determined by the is architecture and the amino acid composition of the binding sites for X and Y.
In the first step of the mechanism, the substrate binds to the enzyme to form the non-covalent Michaelis-Menten complex. In the subsequent step, the covalent intermediate is formed between the enzyme and the acyl moiety of the substrate (E--CO--X). Formation of the acyl-enzyme may occur through cleavage of an amide bond (amide hydrolysis of X--CO--NH--Y) or an ester bond (ester hydrolysis X--CO--O--R) and at low pH it may also be formed directly from X--COOH. The nucleophile YNH binds to the acyl-enzyme before deacylation. Under conditions which favour the deacylation (the enzyme acts as a deacylase or amidase) a water molecule will hydrolyse the acyl enzyme thereby liberating the second product X--COOH and regenerating the enzyme for a new catalytic cycle. Under conditions which favour formation of compound X--CO--NH--Y, the nucleophile Y--NH reacts with the acyl enzyme instead of water (aminolysis). For PenG the mechanism above was confirmed by the observations that phenylacetic acid acts as a competitive inhibitor and 6-APA as a non-competitive one.
In general the formation of the acyl-enzyme from amides (v.sub.1) is slow compared to the hydrolysis of the acyl enzym (v.sub.3). However, when the appropriate ester derivatives of the side chain are used (X--CO--O--R) or just the amide (X--CO--NH2) then the formation of the acyl-enzyme (v.sub.2) is relatively fast in comparison with hydrolysis (v.sub.3). The consequence is that the acyl enzyme intermediate will accumulate. In the presence of suitable compounds with a free primary amino group (general representation Y--NH2) such as, for example, 6-APA, 7-ACA, 7-ADCA which are bound by the acylase, an amide bond may be formed giving X--CO--N--Y (v.sub.-1, aminolysis).
With respect to the preference for chemical entities X and Y substitution of residues in the binding sites for X and Y at the enzyme alter this preference. Changes in substrate specificity include all combinations of increase and decrease of V.sub.sax and K.sub.s. In some cases a more specific enzyme is required, e.g. with mixtures of enantiomers it may be useful when the enzyme is selective for only one of the enantiomers. In other cases, e.g. the conversion of rather pure compounds, a higher conversion rate might be preferred at the cost of selectivety. At high substrate concentrations a higher V.sub.sax is preferred while Km is less important.
Acylases used for substrate activation and kinetically controlled synthesis may be altered in such a way that their catalytic ability to hydrolyse compounds (V.sub.3 =transfer acyl group to water) has been suppressed with respect to acyl transfer to a non-aguous acceptor nucleophile (v.sub.-1): ratio V.sub.-1 /V.sub.3 increased relative to wild type.
The ratio of transferase to hydrolase activity is the enzyme property that influences yield in kinetically controlled synthesis of condensation products. The ratio of the apparent second order rate constants for the acyl transfer to YNH or H20 can be determined from the initial rates of formation of X--CO--NH--Y and X--COOH from the acyl-enzyme.
Transferase activity may be improved by improving the affinity of the nucleophile for the enzyme-acyl complex with respect to water. As the transfer of the acyl group (v.sub.-1) is proportional to amount of nucleophile bound to the acyl-enzyme an increased affinity for the enzyme-acyl complex will improve the yield of the condensation product with respect to hydrolysis.
In addition a higher yield in an enzyme catalysed biosynthesis may be obtained by reducing the hydrolysis of the desired products (v.sub.1 v.sub.3). Variants for which the hydrolysis of amide bonds relative to ester bonds has been decreased are still able to form the acyl enzym from ester substrates (v.sub.2) but have relatively weak hydrolysis activity for the product amide bond (increased ratio v.sub.1 /V.sub.2 with respect to wild type).
Relevant Literature
Several genes encoding Type-IIA Penicillin G acylases have been sequenced, viz. the genes from E. coli (G. Schumacher et al., Nucleic Acids Research 14 (1986) 5713-5727), Kluyvera citrophila (J. L. Barbero et al., Gene 49 (1986) 69-80), Alcaligenes faecalis (U.S. Pat. No. 5,168,048, Gist-brocades), Providencia rettgeri (G. Ljubijankic et al., J. DNA Sequencing and Mapping 3 (1992) 195-201) and Arthrobacter viscosis (M. Konstantinovic et al., (1993) EMBL databank entry L04471).
The use of recombinant DNA methods has enabled an increase of the production levels of commercially used penicillin acylases (Mayer et al., Adv. Biotechnol. 1 (1982) 83-86) and has enlarged the insight into the processing of these enzymes (G. Schumacher et al., Nucleic Acids Research 14 (1986) 5713-5727). The penicillin acylase of E. coli was found to be produced as a large precursor protein, which was further processed into the periplasmic mature protein constituting a small (.alpha.) and a large (.beta.) subunit. Cloning and sequencing of the Kluyvera citrophila acylase gene has revealed a close homology with the E. coli acylase gene (J. L. Barbero et al., Gene 49 (1986) 69-80). Also for Proteus rettaeri (G. O. Daumy et al., J. Bacteriol. 163 (1985) 1279-1281) and Alcaligenes faecalis (U.S. Pat. No. 5,168,048 and EP-A-453048, Gist-brocades) Penicillin G acylase a small and a large subunit has been described.
These publications neither teach nor suggest the instant invention.
Redesigning of specific activity of enzymes with the aid of protein-engineering techniques has been described.
Patent applications EP-A-130756 and EP-A-251446 describe the selection of residues and the mutagenesis of some of these residues in a certain group of serine protease with the purpose to alter the kinetic properties of these enzymes.
As these patent applications specifically deal with a certain type of serine proteases (the subtilisin type), these publications do not indicate which residues modulate the catalytic properties of Type-IIa Penicillin G acylases.
Wells et al. (Proc. Natl. Acad. Sci. USA 84 (1987) 5167) show an example for subtilisin. Bacillus licheniformis and B. amyloliguefaciens serine protease differ by 31% (86 residues) in protein sequence and by a factor of 60 in catalytic efficiency on certain substrates. By substituting 3 of the 86 different amino acids from the B. amyloliguefaciens sequence by the corresponding B. licheniformis residues the catalytic activity of the mutant enzyme was improved nearly 60 fold.
Wilks et al. (Science 242 (1988) 1541) describe how a lactate dehydrogenase was changed into a malate dehydrogenase by mutating glutamine 102 into arginine 102. In both cases, serine protease and lactate dehydrogenase, the inspiration for the modification proposal came from comparison with naturally occuring enzymes, which already showed the desired specificity. In the same way the specificity of cytochrome p450.sub.15.alpha. was changed into the specificity of cytochrome p450.sub.coh by replacing Leu209 with Phe209 (Lindberg and Negishi, Nature 339 (1989) 632).
Patent application WO93/15208 describes a method for modifying the specificity and or efficiency of a dehydrogenase while retaining its catalytic activity, characterized in that it comprises: selecting an enzyme, the tertiary structure of which is substantially known or deduced; identifying at least one specificity and/or efficiency-related region; identifying or constructing unique restriction sites bounding the identified region in the DNA encoding therefor; generating a DNA sequence which corresponds to at least a portion of the identified region, except that the nucleotides of at least one codon are randomized, or selecting as a substitute for at least a portion of the identified region an alternative such region, which may itself be similarly randomized; using the generated or substitute DNA sequence to replace the original sequence; axpressing the DNA including the generated or substitute DNA sequence; and selecting for a desired modification so that the DNA coding therefor may be isolated. As dehydrogenases are in no way related to Penicillin G acylase, this patent application does not reveal the residues in the acylase which should be substituted to alter its kinetic properties.
Forney et al. (Appl. and Environm. Microbiology 55 (1989) 2550-2556; Appl. and Environm. Microbiology 55 (1989) 2556-2560) have isolated by cloning and in vitro chemical/UV random mutagenesis techniques E. coli strains capable of growing on glutaryl-L-leucine or D(-)-.alpha.-amino-phenyl-acetyl-(L)-leucine. Penicillin acylase produced by the mutants hydrolyse glutaryl-L-leucine between pH and 6 or D(-)-.alpha.-amino-phenyl-acetyl-(L)-leucine at pH 6.5. Although it is supposed that the specificity shift of the Penicillin G acylase is due to one or more mutations in the acylase, the residue(s) involved nor the kind of mutation(s) were identified.
J. A. Williams & T. J. Zuzel (Journ. of Cell. Biochem. (1985) supplement 9B, 99) reported in an abstract of a poster presentation the modification of the substrate specificity of Penicillin G acylase by in vitro mutagenesis of a methionine. Although the abstract does not report the position of this methionine, from the poster it seemed to be possible to conclude that it involved position Met168 in E. coli acylase. However, this work did not reveal any details how substitution of this methionine relates to 25 the observed specificity change. Prieto et al. (I. Prieto et al., Appl. Microbiol. Biotechnol. 33 (1990) 553-559) replaced Met168 in K. citrophila for Ala, Val, Asp, Asn, Tyr which affected the kinetic parameters for PenG and PenV deacylation. In addition mutants Lys375Asn and His481Tyr were made which showed hardly any effect on k.sub.cat /Km.
J. Martin et al. analysed mutant Met168Ala in K. citrophila penicillin acylase and reported altered kinetic properties. (J. Martin & I. Prieto, Biochimica et Biophysica Acta 1037 (1990), 133-139). These references indicate the importance of the residue at position 168 in E. coli and K. citrophila for the specificity with respect to the acyl moiety. However, this work did not reveal any details how substitution of this methionine relates to the specificity change for the conversion of a desired substrate.
Wang Min reported mutagenesis of Ser177 in E. coli Penicillin G acylase to Gly, Thr, Leu, Arg but failed to obtain active acylases. (Wang Min et al., Shiyan Shengwu Xuebao hi (1991), 1, 51-54).
Kyeong Sook Choi et al. (J. of Bacteriology 174 (1992) 19, 6270-6276) replaced the .beta.-subunit N-terminal serine in E. coli penicillin acylase by threonine, arginine, glycine and cysteine. Only when the N-terminal residue was cysteine the enzyme was processed properly and a mature enzyme but inactive enzyme was obtained. In addition chemical mutagenesis of the .beta.-subunit N-terminal serine also led to severe/almost complete loss of activity (Slade et al., Eur. J. Biochem. 197 (1991) 75-80; J. Martin et al., Biochem. J. 280 (1991) 659-662).
Sizman et al. (Eur. J. Biochem. 192 (1990) 143-151) substituted serine 838 in E. coli for cysteine without any effect on the post-translational processing nor on the catalytic activity of the enzyme. In addition Sizman et al. made various deletion mutants of penicillin acylase. It showed that correct maturation of the acylase is very sensitive to mutagenesis. All .beta.-subunit C-terminal deletion mutants were not expressed except for the mutant lacking the last three residues which, however, was very unstable. Insertion of four residues in E. coli at position 827 also failed to give active enzyme.
Prieto et al. replaced glycine 310 in Kluyvera citrophila penicillin acylase for glutamic acid. However, no active enzyme was obtained.
In EP-A-453048 it has been described how protein engineering may be used to alter the specificity of Type-IIa as well as Type-IIb acylase. However, the applied procedures are limited to the generation of libraries of randomly generated acylase mutants which have to be screened for a desired activity. Although by the method described in that patent application the number of amino acid positions which may be mutated has been reduced, the number of remaining positions is still large, so that position directed mutagenesis would be a laborious job. The present invention, however, gives a much more limited number of positions which are to be mutated. In addition amino acids at these positions are in direct contact with the substrate, which means that substitution will affect interaction with the substrate directly. Moreover the procedure leading to the present invention allows one to choose a particular amino acid substitution in order to obtain a desired effect for a specific substrate.